Wild coral oocytes are more amenable to low temperature preservation than cultured counterparts.

Few studies have examined the biochemical differences between cultured and wild coral after undergoing low-temperature preservation. The present study aimed to explore the differences in the biochemical characteristics of cultured and wild coral cells and oocytes (Echinopora gemmacea and Oxypora lacera) in cryopreservation conditions. Wild and cultured coral cells were extracted and subjected to freezing experiments involving multiple types and concentrations of cryoprotectant, and the oocytes from the cultured and wild corals were subjected to chilling experiments. Cultured and wild coral cells exhibited no significant differences in viability or cell density after cryopreservation, whereas the oocytes from the cultured corals E. gemmacea and O. lacera exhibited lower chilling tolerance compared with their wild counterparts. Significant differences were observed between the oocytes from the cultured and wild corals after low-temperature preservation, particularly in their metabolic activity and vital status, which could be possibly attributed to food consumption and environmental factors. The study provides a foundation for research promoting the technological development of artificial coral propagation and cryopreservation.

The Antileukemic Effect of Xestoquinone, A Marine-Derived Polycyclic Quinone-Type Metabolite, Is Mediated through ROS-Induced Inhibition of HSP-90.

Xestoquinone is a polycyclic quinone-type metabolite with a reported antitumor effect. We tested the cytotoxic activity of xestoquinone on a series of hematological cancer cell lines. The antileukemic effect of xestoquinone was evaluated in vitro and in vivo. This marine metabolite suppressed the proliferation of Molt-4, K562, and Sup-T1 cells with IC50 values of 2.95 ± 0.21, 6.22 ± 0.21, and 8.58 ± 0.60 µM, respectively, as demonstrated by MTT assay. In the cell-free system, it inhibited the activity of topoisomerase I (Topo I) and II (Topo II) by 50% after treatment with 0.235 and 0.094 µM, respectively. The flow cytometric analysis indicated that the cytotoxic effect of xestoquinone was mediated through the induction of multiple apoptotic pathways in Molt-4 cells. The pretreatment of Molt-4 cells with N-acetyl cysteine (NAC) diminished the disruption of the mitochondrial membrane potential (MMP) and apoptosis, as well as retaining the expression of both Topo I and II. In the nude mice xenograft model, the administration of xestoquinone (1 µg/g) significantly attenuated tumor growth by 31.2% compared with the solvent control. Molecular docking, Western blotting, and thermal shift assay verified the catalytic inhibitory activity of xestoquinone by high binding affinity to HSP-90 and Topo I/II. Our findings indicated that xestoquinone targeted leukemia cancer cells through multiple pathways, suggesting its potential application as an antileukemic drug lead.

13-Acetoxysarcocrassolide Exhibits Cytotoxic Activity Against Oral Cancer Cells Through the Interruption of the Keap1/Nrf2/p62/SQSTM1 Pathway: The Need to Move Beyond Classical Concepts.

13-Acetoxysarcocrassolide (13-AC), a marine cytotoxic product isolated from the alcyonacean coral Lobophytum crassum, exhibited potent antitumor and immunostimulant effects as reported in previous studies. However, the 13-AC antitumor mechanism of action against oral cancer cells remains unclear. The activity of 13-AC against Ca9-22 cancer cells was determined using MTT assay, flow cytometric analysis, immunofluorescence, immunoprecipitation, Western blotting, and siRNA. 13-AC induced apoptosis in oral cancer cells Ca9-22 through the disruption of mitochondrial membrane potential (MMP) and the stimulation of reactive oxygen species (ROS) generation. It increased the expression of apoptosis- and DNA damage-related proteins in a concentration- and time-dependent manner. It exerted potent antitumor effect against oral cancer cells, as demonstrated by the in vivo xenograft animal model. It significantly reduced the tumor volume (55.29%) and tumor weight (90.33%). The pretreatment of Ca9-22 cells with N-acetylcysteine (NAC) inhibited ROS production resulting in the attenuation of the cytotoxic activity of 13-AC. The induction of the Keap1-Nrf2 pathway and the promotion of p62/SQSTM1 were observed in Ca9-22 cells treated with 13-AC. The knockdown of p62 expression by siRNA transfection significantly attenuated the effect of 13-AC on the inhibition of cell viability. Our results indicate that 13-AC exerted its cytotoxic activity through the promotion of ROS generation and the suppression of the antioxidant enzyme activity. The apoptotic effect of 13-AC was found to be mediated through the interruption of the Keap1/Nrf2/p62/SQSTM1 pathway, suggesting its potential future application as an anticancer agent.

Cryopreservation of a Thermotolerant Lineage of the Coral Reef Dinoflagellate Symbiodinium.

Dinoflagellates of the genus Symbiodinium form symbiotic relationships with corals, other marine invertebrates, and protists; thus, they are considered as important species in coral reef ecosystems. If Symbiodinium could be successfully cryopreserved, the cell bank generated could prove to be a valuable resource for researchers interested in basic biological research of Symbiodinium-invertebrate symbioses. Herein, successful cryopreservation of clade D Symbiodinium was achieved using a two-step freezing protocol. Symbiodinium cells were exposed to cryoprotectants (CPAs) for 30 minutes before being vapor frozen for 20 minutes in liquid nitrogen (LN2); afterward, cells were immediately immersed in LN2 for 2 hours or 10 days. The initial experiment was conducted with the following CPAs at 1, 2, and 3 M concentrations: methanol, dimethyl sulfoxide, glycerol, ethylene glycol (EG), and propylene glycol (PG). It was found that infiltration with 2 M EG and PG yielded cells with the highest percentage viability. Upon thawing, culture of these Symbiodinium was carried out for 2 months in a growth chamber, and cells continued to grow and proliferate over this period. This represents successful cryopreservation of a dominant reef coral symbiont, a feat that will ideally aid in future research of this important lineage of dinoflagellate.

In-group comparison is painful but meaningful: The moderator of classroom ethnic composition and the mediators of self-esteem and school belonging for upward comparisons.

This study examined the moderation of classroom ethnic composition and the mediation of group identification in the relationships between upward comparisons, depression, and self-efficacy in a sample of 359 Taiwanese aboriginal students. A stronger negative effect was found in highly ethnically concentrated classes. Upward comparisons were found to reinforce depression, decrease self-esteem, and reduce school belonging in aborigines-only classes but not in mainstream classes. Two pathways-self-esteem and school belonging-were found to process the negative indirect effects on depression and academic and social self-efficacy. The indirect effect through school belonging was particularly strong in the aborigines-only classes compared with the mainstream classes. The suppression effect showed that when self-esteem and school belonging were sustained, the aboriginal students in aborigines-only classes could increase their social self-efficacy through upward comparisons.

Submarine Groundwater Discharge helps making nearshore waters heterotrophic.

Submarine groundwater discharge (SGD) is the submarine seepage of all fluids from coastal sediments into the overlying coastal seas. It has been well documented that the SGD may contribute a great deal of allochthonous nutrients to the coastlines. It is, however, less known how much carbon enters the ocean via the SGD. Nutrients (NO3, NO2, NH4, PO4, SiO2), alkalinity and dissolved inorganic carbon (DIC) in the submarine groundwater were measured at 20 locations around Taiwan for the first time. The total N/P/Si yields from the SGD in Taiwan are respectively 3.28 ± 2.3 × 104, 2.6 ± 1.8 × 102 and 1.89 ± 1.33 × 104 mol/km2/a, compared with 9.5 ± 6.7 × 105 mol/km2/a for alkalinity and 8.8 ± 6.2 × 105 mol/km2/a for DIC. To compare with literature data, yields for the major estuary across the Taiwan Strait (Jiulong River) are comparable except for P which is extremely low. Primary production supported by these nutrient outflows is insufficient to compensate the DIC supplied by the SGD. As a result, the SGD helps making the coastal waters in Taiwan and Jiulong River heterotrophic.

Persistent organic pollutants in Antarctic notothenioid fish and invertebrates associated with trophic levels.

Notothenioid fish and invertebrate samples from Antarctica were collected in the austral summer of 2009, and analyzed for persistent organic pollutants (POPs), including polycyclic aromatic hydrocarbons (PAHs), organochlorine pesticides (OCPs), and polybrominated diphenylethers (PBDEs), as well as δ13C and δ15N stable isotopes for trophic level determination. In this study, the POP levels in the Antarctic biota samples were found to be ranked in the following order: OCPs > PAHs >> PBDEs. The POP levels in notothenioid fish and krill correlate to trophic levels; however, the POP concentrations in intertidal benthic invertebrates are higher than in notothenioid fish implying that specific biogeochemical factors may affect bioaccumulation in the Antarctica ecosystem. Biomagnification of POPs may have a smaller role than bioconcentration in Antarctica environment. In addition to the source, transport, exposure, and absorption for each group of POPs in the short food chain in Antarctica, the biological variation among species, interaction habitats, diet and metabolism are also factors for future studies on contaminant bioaccumulation.

The moderating effect of the need to belong and classroom composition on belongingness seeking of minority adolescents.

This study aimed to elucidate whether the interaction of classroom composition and the need to belong influences belongingness seeking and, if so, to investigate how upward comparison mediates the effects. The analyses were conducted with a cross-sectional sample of 383 Taiwanese aboriginal adolescents (39.7% male) recruited from schools with mixed-sex/ethnicity (n = 113), single-sex (n = 122), and minority-only (n = 148) classrooms. After controlling for socioeconomic status, the moderation analyses indicated that participants with a chronic need to belong in classes with diversity (mixed sex/ethnicity) perceived higher social acceptance, while those with a chronic need to belong in homogeneous classes (single-sex and minority-only) reported greater feelings of rejection. Upward comparison for differentiation was found to influence the indirect effects of the need to belong on feelings of rejection and depression in single-sex and minority-only classes. In particular, the mediating effect of upward comparison was stronger in minority-only classes.

Unveiling the transformation and bioavailability of dissolved organic matter in contrasting hydrothermal vents using fluorescence EEM-PARAFAC.

The submarine hydrothermal systems are extreme environments where active cycling of dissolved organic matter (DOM) may occur. However, little is known about the optical properties and bioavailability of hydrothermal DOM, which could provide valuable insights into its transformation processes and biogeochemical reactivity. The quantity, quality, and bioavailability of DOM were investigated for four very different hydrothermal vents east of Taiwan, using dissolved organic carbon (DOC), absorption spectroscopy, and fluorescence excitation-emission matrices-parallel factor analysis (EEM-PARAFAC). The DOC and absorption coefficient a280 were both lower in the two hydrothermal vents off the Orchid Island and on the Green Island than in the surrounding seawater and the two vents off the Kueishantao Island, indicating effective removals of DOM in the former two hydrothermal systems owing to possible adsorption/co-precipitation and thermal degradation respectively. The four hydrothermal DOM showed notable differences in the absorption spectral slope S275-295, humification index HIX, biological index BIX, EEM spectra, and the relative distributions of seven PARAFAC components. The results demonstrated a high diversity of chemical composition and transformation history of DOM under contrasting hydrothermal conditions. The little change in the hydrothermal DOC after 28-day microbial incubations indicated a low bioavailability of the bulk DOM, and different PARAFAC components showed contrasting bioavailability. The results have profound implications for understanding the biogeochemical cycling and environmental effects of hydrothermal DOM in the marine environments.

Validation of reference genes for cryopreservation studies with the gorgonian coral endosymbiont Symbiodinium.

Quantification by real-time RT-PCR requires a stable internal reference known as a housekeeping gene (HKG) for normalising the mRNA levels of target genes. The present study identified and validated stably expressed HKGs in post-thaw Symbiodinium clade G. Six potential HKGs, namely, pcna, gapdh, 18S rRNA, hsp90, rbcl, and ps1, were analysed using three different algorithms, namely, GeNorm, NormFinder, and BestKeeper. The GeNorm algorithm ranked the candidate genes as follows in the order of decreasing stability: pcna and gapdh > ps1 > 18S rRNA > hsp90 > rbcl. Results obtained using the NormFinder algorithm also showed that pcna was the most stable HKG and ps1 was the second most stable HKG. We found that the candidate HKGs examined in this study showed variable stability with respect to the three algorithms. These results indicated that both pcna and ps1 were suitable for normalising target gene expression determined by performing real-time RT-PCR in cryopreservation studies on Symbiodinium clade G. The results of the present study would help future studies to elucidate the effect of cryopreservation on gene expression in dinoflagellates.

The complete mitochondrial genome of Montipora aequituberculata (Scleractinia, Acroporidae).

The complete mitogenome of the hexacorallia, Montipora aequituberculata has been amplified and sequenced. The mitogenome consists of 17,886 bp, with 13 protein-coding genes, 2 ribosomal RNA genes, 2 transfer RNA genes and a control region. It has been observed that ND5 gene is split into two parts by a large fragment of genes, which commonly presented in scleractinian coral. The overall base composition of the H-strand was A, 24.91%; G, 24.1%; C, 14.2%; and T, 36.8%, with a slight AT bias of 61.7%. The control region was 627 bp in length and located between 12S rRNA and COIII gene. Based on the neighbour-joining (NJ) tree, M. aequituberculata was grouped with M. cactus, Anacropora matthai and Acropora tenuis, and formed a clade of Acroporidae. In conclusion, the complete mitogenome of M. aequituberculata data may provide more informative for phylogenetic approach for corals phylogeny.

Impacts of low temperature preservation on mitochondrial DNA copy number in oocytes of the hard coral Echinopora sp.

Given the current threats to coral reefs worldwide, there is an urgent need to develop protocols for the cryopreservation of reef-building corals. However, chilling may alter coral mitochondrial distribution and membrane potential, resulting in reduced ATP production. The aim of this study was to investigate the impacts of chilling on mitochondrial DNA copy number (CN) in oocytes of the hard coral Echinopora sp. Oocytes were exposed to 0.5 M, 1 M or 2 M methanol at 5, 0 or -5 °C for 2, 4, 8 and 16 h. When oocytes were chilled with no cryoprotectant (CPT) or 1 M methanol at 5 or 0 °C, the mtDNA CNs initially increased at hour 2 of incubation, although it decreased significantly over the 16 h of incubation in chilled oocytes at -5 °C. The mtDNA CN increased and picked in 0.5 M methanol at 5 °C and 0 °C at hour 8 of incubation in chilled oocytes indicating that the high mtDNA CN of these oocytes is probably responsible for withstanding high chilling sensitivity. We currently propose that 0.5 M methanol is the optimal CPT for oocytes of Echinopora sp., and potentially other reef corals.

Membrane lipid phase transition behavior of oocytes from three gorgonian corals in relation to chilling injury.

The lipid phase transition (LPT) from the fluid liquid crystalline phase to the more rigid gel structure phase that occurs upon exposure to low temperatures can affect physical structure and function of cellular membranes. This study set out to investigate the membrane phase behavior of oocytes of three gorgonian corals; Junceela fragilis, J. juncea and Ellisella robusta,at different developmental stages after exposure to reduced temperatures. Oocytes were chilled to 5°C for 48, 96 or 144 h, and the LPT temperature (LPTT) was determined with Fourier Transform Infrared (FTIR) spectroscopy. The J. fragilis oocytes had a higher LPTT (∼23.0-23.7°C) than those of J. juncea and E. robusta oocytes (approximately 18.3-20.3°C). Upon chilling for 96 h at 5°C, the LPTTs of J. juncea and E. robusta oocytes in the early (18.0±1.0 and 18.3±0.6°C, respectively) and late (17.3±0.6 and 17.7±1.2°C, respectively) stages were significantly lower than those of J. fragilis oocytes (20.3±2.1 and 19.3±1.5°C for the early and late stages, respectively). The LPTTs of early stage gorgonian oocytes was significantly lower than those of late stage oocytes. These results suggest that the LPT of three gorgonian oocytes at different developmental stages may have been influenced by the phospholipid composition of their plasma membranes, which could have implications for their low temperature resistance.

10-acetylirciformonin B, a sponge furanoterpenoid, induces DNA damage and apoptosis in leukemia cells.

10-Acetylirciformonin B, a furanoterpenoid derived from irciformonin B found in a marine sponge, has been reported to possess potent cytotoxic activity against several cancer cell lines. However, the mechanism of its apoptotic activity against human leukemia cells has never been reported. The purpose of this study was to investigate the cytotoxic effects of 10-acetylirciformonin B and its possible mechanism of action against leukemia HL 60 cells. We found that 10-acetylirciformonin B decreased cell viability through the inhibition of cell growth as well as the induction of DNA damage and apoptosis in a dose-dependent manner. The induction of DNA damage was mediated by the increase of p-CHK2 and γ-H2A.X, which was suggested from the increase of tail movement in the neutral Comet assay. Induction of apoptosis was mediated with the increase in caspases 8, 9 and 3 activation as well as PARP cleavage. In summary, our resultsindicate that 10-acetylirciformonin B treatment causes apoptosis in leukaemia cells; probably through a caspase-dependent regulatory pathway.

Isolation of marine bacteria with antimicrobial activities from cultured and field-collected soft corals.

Bacteria associated with eight field-collected and five cultured soft corals of Briareum sp., Sinularia sp., Sarcophyton sp., Nephtheidae sp., and Lobophytum sp. were screened for their abilities in producing antimicrobial metabolites. Field-collected coral samples were collected from Nanwan Bay in southern Taiwan. Cultured corals were collected from the cultivating tank at National Museum of Marine Biology and Aquarium. A total of 1,526 and 1,138 culturable, heterotrophic bacteria were isolated from wild and cultured corals, respectively; seawater requirement and antimicrobial activity were then assessed. There is no significant difference between the ratio of seawater-requiring bacteria on the wild and cultured corals. The ratio of antibiotic-producing bacteria within the seawater-requiring bacteria did not differ between the corals. Nineteen bacterial strains that showed high antimicrobial activity were selected for 16S rDNA sequencing. Three strains could be assigned at the family level (Rhodobacteraceae). The remaining 16 strains belong to eight genera: Marinobacterium (2 strains), Pseudoalteromonas (1), Vibrio (5), Enterovibrio (1), Tateyamaria (1), Labrenzia (2), and Pseudovibrio (4). The crude extract from bacteria strains CGH2XX was found to have high cytotoxicity against the cancer cell line HL-60 (IC(50) = 0.94 µg/ml) and CCRF-CEM (IC(50) = 1.19 µg/ml). Our results demonstrate that the marine bacteria from corals have great potential in the discovery of useful medical molecules.

Lipid content and composition during the oocyte development of two gorgonian coral species in relation to low temperature preservation.

Our previous studies have suggested that chilling sensitivity of coral oocytes may relate to their relatively high lipid intracellular content and lipid composition. The distribution of lipids during the oocyte development was determined here for the first time in two gorgonian species (Junceella juncea and Junceella fragilis). The main lipid classes in the two gorgonian oocytes were total lipid, wax ester, triacylglycerol, total fatty acid, phosphatidylethanolamine and phosphatidylcholine. The results indicated that early stage oocytes of J. juncea and J. fragilis were found to have increased lipid content than late stage oocytes. The content of wax ester was significantly higher in the early stage oocytes of two gorgonian corals (51.0±2.5 and 41.7±2.9 µg/mm(3)/oocyte) than those of late stage oocytes (24.0±1.4 and 30.4±1.2 µg/mm(3)/oocyte, respectively). A substantial amount of phosphatidylethanolamine and total fatty acid was detected at each stage of oocyte development in two gorgonian ranges from 107 to 42 µg/mm(3)/oocyte and 106 to 48 µg/mm(3)/oocyte, whilst low levels of phosphatidylcholine were found in two gorgonian oocytes. The levels of total lipid in the late stage oocytes of J. juncea were significantly higher than those of J. fragilis. The observed differences may partially be related to different habitat preferences as higher lipid levels in J. juncea, a deeper-water coral species exposed to lower temperature seawater, might relate to adjustments of cell membranes in order to increase membrane fluidity.